Project Summary Nucleosomes represent a mechanical and energetic barrier to transcription by eukaryotic RNA polymerases. The dynamics modulation of this barrier in the cell is a major mechanism of gene expression regulation. Improper regulation of the nucleosomal barrier results in numerous pathological conditions, including cancer. Here, we will use high resolution optical tweezers with single molecule fluorescence detection (?fleezers?) to characterize the modulation of transcriptional dynamics by nucleosomes, and how the human Pol II (hPol II) affects nucleosome integrity. We will first characterize the elongation dynamics of single hPol II. Specifically, we will follow the progress of hPol II at single base pair (bp) resolution and at a position accuracy of 3 bp. We will measure the pause-free velocity, the pausing probability, pause duration, and backtracking dynamics of hPol II, and test how these dynamics are modulated by factors such as force, elongation factors, the phosphorylation state of the C-terminal domain of RPB1, as well as the presence of torsional constrains on the template DNA. This analysis will results in a detailed description of the mechanochemical cycle of hPol II and how it is regulated. In parallel, we will characterize the energetics and dynamics of the nucleosomal barrier using two approaches: 1) mechanically unwrapping the DNA from the surface of the histone octamer and 2) mechanically unzipping the strands of the DNA sequentially around the octamer. We will investigate how the barrier is modulated by histone variants and epigenetic modifications that appear in +1 nucleosomes (H2A.Z, H3K9ac and ubiquitinated H2B) or inside gene bodies (H3K36me3 and H3K79me3). Importantly, we will also combine these force-extension measurements with detection of fluorescently labelled histone components of the octamer (using a newly built ?fleezers? system) to establish the structural changes that occur in the nucleosome during mechanical unwrapping and unzipping of the DNA. We seek to obtain a detailed description of the height, depth, and symmetry of the barrier and its alteration by epigenetic modifications. Next, we will establish how the nucleosomal barrier modifies the dynamics of hPol II and, in turn, what is the effect of the transcribing enzyme on the integrity of nucleosomes using the fleezers system. We will investigate how epigenetic modifications of the barrier, the topological constraint of the template, and elongation factors alter the dynamics of hPol II and how they affect the stability of the barrier to the passage of the enzyme. In collaboration with Prof. Xavier Darzacq, we will compare the dynamics of hPol II obtained in- vitro with those observed in-vivo in the context of nucleosomes, by tracking the fluorescence of tandem repeats of MS2 bacteriophage RNA binding domains in U2OS cells. We will also perform ex-vivo experiments using nuclear extracts. We hope to obtain an unprecedented quantitative description of the physical/physiological mechanisms that control gene expression. 1